JP2728148B2 - Separation of aminoarginine and uses to block nitric oxide production in the body - Google Patents

Separation of aminoarginine and uses to block nitric oxide production in the body

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Publication number
JP2728148B2
JP2728148B2 JP2510618A JP51061890A JP2728148B2 JP 2728148 B2 JP2728148 B2 JP 2728148B2 JP 2510618 A JP2510618 A JP 2510618A JP 51061890 A JP51061890 A JP 51061890A JP 2728148 B2 JP2728148 B2 JP 2728148B2
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Prior art keywords
arginine
amino
nitric oxide
aminoarginine
nitro
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JPH05500659A (en
Inventor
グリフィス、オーウェン・タブリュ
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Cornell Research Foundation Inc
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Cornell Research Foundation Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/26—Androgens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C277/00—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C277/08—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine

Abstract

Pharmaceutically pure physiologically active N<G>-aminoarginine (i.e., the L or D,L form) or pharmaceutically acceptable salt thereof is administered in a nitric oxide synthesis inhibiting amount to a subject in need of such inhibition (e.g. a subject with low blood pressure or needing immunosuppressive effect) or is added to a medium containing isolated organs, intact cells, cell homogenates or tissue homogenates in an amount sufficient to inhibit nitric oxide formation to elucide or control the biosynthesis, metabolism or physiological role of nitric oxide. N<G>-amino-L-arginine is prepared and isolated as a pharmaceutically pure compound by reducing N<G>-nitro-L-arginine, converting L-arginine by-product to L-ornithine with arginase and separating N<G>-amino-L-arginine from the L-ornithine. N<G>-amino-D,L-arginine is prepared in similar fashion starting with N<G>-nitro-D,L-arginine.

Description

【発明の詳細な説明】 本発明は、少くとも一部分はアメリカ予防衛生研究所
認可番号DK37116の下で米国政府の援助によりなされた
ものである。米国政府は本発明に一定の権利を有するも
のである。DETAILED DESCRIPTION OF THE INVENTION This invention was made, at least in part, with the support of the United States Government under the U.S.A. The United States government has certain rights in the invention.

技術分野 本発明は生物学的酸化窒素生成の新規な阻害剤に関す
る。TECHNICAL FIELD The present invention relates to novel inhibitors of biological nitric oxide production.

発明の背景 数10年来ニトログリセリンは心臓血管病の処置におけ
る血管拡張剤としてヒトに投与されてきた。最近、その
ように投与されたニトログリセリンが医薬的に活性な代
謝物である酸化窒素に身体内で変換されることが示され
た。さらに極く最近酸化窒素は内皮由来の弛緩因子（ED
RF類）の重要成分である正常な代謝物としてアルギニン
から生成されることが示された。EDRF類は現在血流およ
び血管抵抗の調節に関与することについて集中的に研究
されている。上記の研究に付随して、探索が身体内の酸
化窒素を遮断する化合物について行われた。この作用を
得るのに用いるために発見された化合物はNG−メチル−
Ｌ−アルギニンである［パルマー，アール．エム．ジェ
イ他、ネーチャー（ロンドン）、第333巻、664−666
頁、1988年］。モルモットおよびウサギへのNG−メチル
−Ｌ−アルギニンの投与は血圧を増大することが示され
た（アイサカ、ケイ他、バイオケミカル・アンド・バイ
オフィジイク・リサーチ・コミュニケーション、第160
巻、２号、881−886頁、1989年４月28日；ニース、デイ
ー．デイー他、プロシーディングス・オブ・ザ・ナショ
ナル・アカデミー・オブ・サイエンス・ユーエスエー、
第86巻、3375−3378頁、1989年５月）。BACKGROUND OF THE INVENTION For decades nitroglycerin has been administered to humans as a vasodilator in the treatment of cardiovascular disease. Recently, it has been shown that nitroglycerin so administered is converted in the body to nitric oxide, a pharmaceutically active metabolite. More recently, nitric oxide has been associated with endothelium-derived relaxing factor (ED
It has been shown to be produced from arginine as a normal metabolite, a key component of RFs. EDRFs are currently being intensively studied for their involvement in regulating blood flow and vascular resistance. Accompanying the above study, a search was conducted for compounds that block nitric oxide in the body. The compound discovered for use in obtaining this effect is NG -methyl-
L-arginine [Palmer, Earl. M. Jay et al., Nature (London), Volume 333, 664-666
P. 1988]. Administration of NG -methyl-L-arginine to guinea pigs and rabbits has been shown to increase blood pressure (Aisaka, Kei et al., Biochemical and Biophysics Research Communication, No. 160).
Vol. 2, No. 881-886, April 28, 1989; Nice, Day. Dee and others, Proceedings of the National Academy of Science USA,
86, 3375-3378, May 1989).

血管内皮以外に、マクノファージはまた、それらの細
胞死滅および／または細胞増殖抑制機能の成分である酸
化窒素を身体内で生成することが示された（イエンガ
ー、アール他、プロシーディングス・オブ・ザ・ナショ
ナル・アカデミー・オブ・サイエンス・ユーエスエー、
第84巻、6373−6373頁、1987年９月）。In addition to vascular endothelium, macnophages have also been shown to produce nitric oxide in the body, a component of their cell killing and / or cytostatic functions (Jengar, R. et al., Proceedings of the. National Academy of Science USA,
84, 6373-6373, September 1987).

発明の概要 生理学的に活性なNG−アミノアルギニン（ここで用語
NGはグアニジノ窒素での置換を示す）およびその医薬的
に許容可能な酸付加塩が身体内の酸化窒素合成のすぐれ
た阻害剤を構成することが本発明により発見された。用
語「生理学的に活性なNG−アミノアルギニン」は本明細
書でNG−アミノ−Ｌ−アルギニンおよびNG−アミノ−D,
L−アルギニンからなる群より選ばれたNG−アミノアル
ギニンを意味するのに用いられる。D,L−化合物におい
て、NG−アミノ−Ｌ−アルギニン部分のみが生理学的に
活性である。SUMMARY OF THE INVENTION Physiologically active NG -aminoarginine (herein the term
NG indicates substitution with guanidino nitrogen) and its pharmaceutically acceptable acid addition salts constitute a good inhibitor of nitric oxide synthesis in the body. The term "physiologically active NG -aminoarginine" is used herein for NG -amino-L-arginine and NG -amino-D,
Used to mean NG -aminoarginine selected from the group consisting of L-arginine. In the D, L-compound, only the NG -amino-L-arginine moiety is physiologically active.

NG−アミノ−Ｌ−アルギニンはペプチドの化学的合成
に用いられるNG−ニトロ−Ｌ−アルギニンの還元的脱保
護における副産物として報告された。しかし、上記のも
のまたは医薬的に純粋な形でのNG−アミノ−D,L−アル
ギニンの製造および分離についての報告は知られていな
い。ここに記載した本発明は上記の製造および分離を意
図している。 NG -amino-L-arginine has been reported as a by-product in the reductive deprotection of NG -nitro-L-arginine used in chemical synthesis of peptides. However, no reports are known on the production or isolation of NG -amino-D, L-arginine as described above or in pharmaceutically pure form. The invention described herein is intended for the above-described production and separation.

すなわち、新規であると考えられる本発明の組成物
は、医薬的に純粋な生理学的に活性なNG−アミノアルギ
ニンまたはその医薬的に許容可能な酸付加塩である。用
語「医薬的に純粋な」は99.9＋％純度（水を除いた成分
での）意味するために本明細書で用いられる。That is, the compositions of the present invention that are considered to be novel are pharmaceutically pure physiologically active NG -aminoarginine or a pharmaceutically acceptable acid addition salt thereof. The term “pharmaceutically pure” is used herein to mean 99.9 +% pure (with the exception of water).

生理学的に活性なNG−アミノアルギニンの製造および
分離のためのここに記載した本発明の方法は：（ａ）NG
−ニトロ−Ｌ−アルギニンまたはNG−ニトロ−D,L−ア
ルギニンを還元して生理学的に活性なNG−アミノアルギ
ニンおよびアルギニンの混合物を生成すること；（ｂ）
アルギナーゼにより上記の混合物を処理してその中のア
ルギニンをオルニチンに変換し生理学的に活性なNG−ア
ミノアルギニンおよびオルニチンの混合物を生成するこ
と；（ｃ）段階（ｂ）により生成される混合物から医薬
的に純粋で生理学的に活性なNG−アミノアルギニンを分
離することの段階を含む。The process of the invention described herein for the preparation and separation of physiologically active NG -aminoarginine comprises: (a) NG
Reducing nitro-L-arginine or NG -nitro-D, L-arginine to produce a mixture of physiologically active NG -aminoarginine and arginine; (b)
Treating said mixture with arginase to convert arginine therein to ornithine to produce a mixture of physiologically active NG -aminoarginine and ornithine; (c) from the mixture produced by step (b) Separating the pharmaceutically pure and physiologically active NG -aminoarginine.

段階（ｃ）の分離は、クロマトグラフィによるかまた
はフラビアン酸塩としてのNG−アミノアルギニンの結晶
化によって簡単に行われる。The separation of step (c) is carried out simply by chromatography or by crystallization of NG- aminoarginine as a flavinate.

阻害を必要とする対象における酸化窒素合成の阻害の
ための本明細書に記載する方法は、上記の対象に酸化窒
素合成を阻害する量の生理学的に活性なNG−アミノアル
ギニンまたはその医薬的に許容可能な酸付加塩を投与す
ることを含む。用語「対象」はアルギニンからの酸化窒
素生成が起るような、ヒトを含んでいる全ての哺乳動物
を意味するために本明細書で用いられる。この方法は予
防および治療の用途を意図している。The method described herein for inhibiting nitric oxide synthesis in a subject in need thereof comprises administering to said subject an amount of a physiologically active NG -aminoarginine or a pharmaceutically acceptable salt thereof that inhibits nitric oxide synthesis. Administering an acceptable acid addition salt. The term "subject" is used herein to mean any mammal, including humans, where nitric oxide production from arginine occurs. This method is intended for prophylactic and therapeutic uses.

酸化窒素の生合成、代謝または生理学的役割を解明す
るかまたは制御するための、分離した器官、無傷の細
胞、細胞ホモジェネートおよび組織ホモジェネートによ
る研究を含めたイン ビトロの研究におけるアルギニン
からの酸化窒素生成の遮断のための本発明の方法は、上
記の器官、細胞、またはホモジェネートを含んでいる培
地に生理学的に活性なNG−アミノアルギニンまたはその
医薬的に許容可能な酸付加塩を酸化窒素生成を阻害する
のに充分な濃度で添加することを含む。Nitric oxide production from arginine in in vitro studies, including studies with isolated organs, intact cells, cell and tissue homogenates, to elucidate or control the biosynthesis, metabolism or physiological role of nitric oxide The method of the present invention for the blockade of comprises converting a physiologically active NG -aminoarginine or a pharmaceutically acceptable acid addition salt thereof into nitric oxide to a medium containing the organ, cell or homogenate described above. And adding at a concentration sufficient to inhibit the activity of

詳細な説明 先に示したように、本発明の組成物は医薬的に純粋で
生理学的に活性なNG−アミノアルギニンまたはその医薬
的に許容可能な酸付加塩が構成する。DETAILED DESCRIPTION As indicated above, the compositions of the present invention comprise a pharmaceutically pure and physiologically active NG -aminoarginine or a pharmaceutically acceptable acid addition salt thereof.

遊離塩基形のNG−アミノ−Ｌ−アルギニンは下記構造
式を有している。The free base form of NG -amino-L-arginine has the following structural formula:

遊離塩基形のNG−アミノ−D,L−アルギニンは50％NG
−アミノ−Ｌ−アルギニンおよび下記構造式を有してい
る50％NG−アミノ−Ｄ−アルギニンから成っている。 NG -amino-D, L-arginine in free base form is 50% NG
-Amino-L-arginine and 50% NG -amino-D-arginine having the following structural formula:

医薬的に許容可能な酸付加塩は高いpkaをもつ窒素に
おいて最初に生成し、たとえば塩酸、硫酸、酢酸、グル
コン酸、リン酸、コハク酸、マレイン酸およびクエン酸
付加塩を包含する。それらは当業界では周知の方法によ
り生成される。 Pharmaceutically acceptable acid addition salts are formed first at nitrogen with a high pka and include, for example, hydrochloric, sulfuric, acetic, gluconic, phosphoric, succinic, maleic and citrate addition salts. They are produced by methods well known in the art.

次に医薬的に純粋で生理学的に活性なNG−アミノアル
ギニンを製造し、分離する方法を述べる。NG−ニトロア
ルギニン出発物質は、NG−アミノ−Ｌ−アルギニンの製
造の場合NG−ニトロ−Ｌ−アルギニンであり、NG−アミ
ノ−D,L−アルギニンの製造の場合NG−ニトロ−D,L−ア
ルギニンである。NG−ニトロ−Ｌ−アルギニンは既に市
販されている。NG−ニトロ−D,L−アルギニンはD,L−原
料から出発する点を除いてNG−ニトロ−Ｌ−アルギニン
と同じ方法で製造される。NG−ニトロ−Ｌ−アルギニン
が出発物質である場合、段階（ａ）の生成物はNG−アミ
ノ−Ｌ−アルギニンおよびＬ−アルギニンの混合物であ
る。NG−ニトロアルギニンの還元は適当な還元触媒（た
とえば、白金炭素、酸化白金、パラジウム炭素および還
元方法に周知の他の触媒）の存在下で過剰の水素ガスを
用いる溶液中の反応により容易に行われる。適当な溶媒
は水性酸、たとえば15％水性酢酸である。還元反応は室
温で行われるが、たとえば０℃−100℃またはより高い
範囲内にある温度が用いられ得る。加減反応は40ポンド
／平方インチの圧力で容易に進むが、１−2000ポンド／
平方インチの範囲内にある圧力で行われ得る。Next, a method for producing and separating pharmaceutically pure and physiologically active NG -aminoarginine will be described. N G - nitro-arginine starting material, N G - For the production of amino -L- arginine N G - nitro -L- arginine, N G - amino -D, when the production of L- arginine N G - nitro -D, L-arginine. NG -nitro-L-arginine is already commercially available. NG -nitro-D, L-arginine is prepared in the same manner as NG -nitro-L-arginine except starting from the D, L-raw material. When NG -nitro-L-arginine is the starting material, the product of step (a) is a mixture of NG -amino-L-arginine and L-arginine. Reduction of NG -nitroarginine is facilitated by reaction in solution with excess hydrogen gas in the presence of a suitable reducing catalyst (e.g., platinum on carbon, platinum oxide, palladium on carbon and other catalysts well known in reduction processes). Done. A suitable solvent is an aqueous acid, such as 15% aqueous acetic acid. The reduction reaction is performed at room temperature, but temperatures in the range of, for example, 0 ° C-100 ° C or higher may be used. The moderation reaction easily proceeds at a pressure of 40 pounds per square inch, but 1-2000 pounds per square inch.
It can be performed at pressures in the range of square inches.

段階（ｂ）において、Ｌ−アルギニンはＬ−オルニチ
ンに変換されるか、またはD,L−アルギニンはD,L−オル
ニチンに変換される。段階（ｂ）のため、段階（ａ）の
触媒は、たとえば濾過することにより除去され、溶媒
は、たとえば減圧下ロータリー蒸発により除去される。
生成する残留物は水に溶解され、わずかにアルカリpH、
たとえば塩基たとえばNaOHの添加による８−10の範囲内
にあるpHに調節される。次にアルギナーゼ（市販品を容
易に入手できる）が、たとえば0.5−5mg/NG−ニトロア
ルギニン出発物質ｇの範囲内にある濃度で添加される。
反応は、たとえば20℃−40℃で８−12時間、好ましくは
37℃で一晩中の培養により行われる。反応の終了後にア
ルギナーゼはたとえば熱変性および遠心分離または濾過
により除去され、生成した生成物は段階（ｃ）に付され
る。In step (b), L-arginine is converted to L-ornithine or D, L-arginine is converted to D, L-ornithine. For step (b), the catalyst of step (a) is removed, for example, by filtration, and the solvent is removed, for example, by rotary evaporation under reduced pressure.
The resulting residue is dissolved in water, slightly alkaline pH,
The pH is adjusted to a value in the range of 8-10, for example, by the addition of a base such as NaOH. Then arginase (commercially readily available), for example 0.5-5 mg / N G - is added at a concentration which is within the range of nitroarginine starting material g.
The reaction is carried out, for example, at 20 ° C.-40 ° C. for 8-12 hours, preferably
Performed by overnight culture at 37 ° C. After the end of the reaction, the arginase is removed, for example by heat denaturation and centrifugation or filtration, and the product formed is subjected to step (c).

段階（ｃ）のクロマトグラフィ分離は強酸性陽イオン
交換樹脂（たとえば、水素またはナトリウム塩の形での
ダウェックス50）および中程度から強い塩基性溶液、た
とえば水と希水酸化アンモニウムとの間に形成される勾
配による溶出により容易に行われる。画分は集められ、
高速液体クロマトグラフィ（以下HPLC）により分析され
る。適当な画分はプールされ、オルニチン−およびアル
ギニン−を欠く生成物がたとえばロータリー蒸発により
分離される。The chromatographic separation of step (c) is formed between a strongly acidic cation exchange resin (eg, Dowex 50 in the form of hydrogen or sodium salt) and a moderate to strong basic solution, eg, between water and dilute ammonium hydroxide. This is easily accomplished by elution with a gradient. Fractions are collected,
It is analyzed by high performance liquid chromatography (hereinafter HPLC). Appropriate fractions are pooled and the products lacking ornithine and arginine are separated off, for example by rotary evaporation.

フラビアン酸塩としてのNG−アミノアルギニンの結晶
化を含んでいる段階（ｃ）の実施態様について述べる
と、これは、pHが約3.8になるまで、フラビアン酸（た
とえば、６−10gms/100ml）の水溶液を添加すること、
たとえば２−10℃に冷却することおよび、たとえば濾過
により生成される黄色沈澱物を集めることにより行われ
る。沈澱物は好ましくNaOHおよびHClにより連続的にpH
を上げ下げすることにより再晶出され、医薬的に純粋な
NG−アミノアルギニンの溶液は、再結晶したフラビアン
酸塩から、強塩基陰イオン交換樹脂（たとえば、ダウェ
ックス50）の懸濁物と共に上記の塩を撹拌して、フラビ
アン酸を結合し、NG−アミノアルギニンを放出すること
により製造される。フラビアン酸を結合した樹脂は、た
とえば濾過することにより分離される。医薬的に純粋で
生理学的に活性なNG−アミノアルギニン生成物は蒸発す
ることまたは他の方法で濾液を乾燥することにより固体
として回収される。N G as flavianic salt - To describe the implementation of step (c) comprising the crystallization of amino arginine, which, until the pH is about 3.8, flavianic acid (e.g., 6-10gms / 100ml) Adding an aqueous solution of
For example, by cooling to 2-10 ° C and collecting the yellow precipitate formed, for example, by filtration. The precipitate is preferably continuously pH adjusted with NaOH and HCl.
Is recrystallized by raising and lowering
N G - amino arginine solution, the recrystallized flavianic acid salt, a strong base anion exchange resins (e.g., Dowex 50) by stirring the salt with suspension, bind the flavianic acid, N G -Produced by releasing aminoarginine. The resin to which the flavonic acid is bound is separated, for example, by filtration. The pharmaceutically pure, physiologically active NG -aminoarginine product is recovered as a solid by evaporation or otherwise drying the filtrate.

次に、上記の阻害を必要とする対象に酸化窒素合成を
阻害する量の生理学的に活性なNG−アミノアルギニンま
たはその酸付加塩を投与することを含む発明のイン ビ
ボの方法を述べる。Next, an in vivo method of the invention comprising administering a physiologically active NG -aminoarginine or an acid addition salt thereof in an amount that inhibits nitric oxide synthesis to a subject in need of such inhibition is described.

対象の１群は病理学的に低血圧をもつものを含む。 One group of subjects includes those with pathologically low blood pressure.

この群の中の１組は自然発症的低血圧症をもつもので
ある。One set in this group has spontaneous hypotension.

この群の中の他の組は薬剤誘発低血圧症をもつもので
ある。この場合に、本発明の方法に従った併用は、そう
でなければ許容不可能な副作用をもつ薬剤の使用を可能
にする。The other set in this group has drug-induced hypotension. In this case, the combination according to the method of the invention allows the use of drugs with otherwise unacceptable side effects.

さらにこの群の中の他の組はショック（毒物ショック
症候群を包含している）にかかっているものである。Yet another group in this group is those who are shocked (including toxic shock syndrome).

速達便により1989年９月13日に提出した“インヒビシ
ョン・オブ・システミック・ハイポテンション・プロデ
ュースド・バイ・バイオロジック・レスポンス・モデフ
ァイアズ”と題するロバート・ジー・キルボルン、ステ
ィーブン・エス・グロス、オウエン・ダブリュー・グリ
フィスおよびロバート・レヴィの出願を引用する。キル
ボルンらの出願は本明細書で請求したものを含めた化合
物の全身性低血圧症阻害用途を包含する。Robert G. Kilbourne and Steven S., "Inhibition of Systemic High Potential Produced by Biologic Response Modifiers" filed September 13, 1989 by express delivery Reference the applications of Gross, Owen W. Griffith and Robert Levy. The application of Kilbourn et al. Includes the use of compounds, including those claimed herein, in inhibiting systemic hypotension.

対象の他の群は、酸化窒素生成の下向き調節が有用で
ある免疫障害患者、たとえば自己免疫障害患者、または
移植を目的とする治療的免疫抑制患者を含む。Other groups of subjects include immunocompromised patients where down regulation of nitric oxide production is useful, for example, autoimmune compromised patients, or therapeutic immunosuppressed patients for transplantation.

次に投与用量を述べると、これは目的とする効果およ
び個々の対象の敏感性により異なる。たとえば、血圧を
上昇させるには、血圧を効果的に上昇させる量が投与さ
れる。免疫抑制を必要とする疾患には、免疫抑制に有効
な量が投与される。一般に、10マイクログラム/kg−100
mg/kg、好ましくは１−10mg/kgの用量範囲が有用であ
る。NG−アミノ−D,L−アルギニンについては、用量はN
G−アミノ−Ｌ−アルギニンについての倍である。Turning now to the dosages to be administered, this depends on the desired effect and the sensitivity of the individual subject. For example, to increase blood pressure, an amount that effectively increases blood pressure is administered. For diseases requiring immunosuppression, an immunosuppressive amount is administered. Generally, 10 micrograms / kg-100
A dose range of mg / kg, preferably 1-10 mg / kg, is useful. For NG -amino-D, L-arginine, the dose is N
Folds for G -amino-L-arginine.

投与は、たとえば経口または非経口経路により容易に
行われる。Administration is facilitated, for example, by the oral or parenteral route.

生理学的に活性なNG−アミノアルギニンは代表的な賦
形剤、香料等と併用して容易に投与される。Physiologically active NG -aminoarginine is easily administered in combination with typical excipients, flavors and the like.

以下本明細書でのイン ビトロの方法を述べる。代表
的な媒質は心臓かん流媒質、組織培養媒質、細胞または
組織のホモジェネートと共に用いられる培養媒質または
精製蛋白質を包含する。処置する器官は代表的に血管、
肺臓または腎臓である。無傷の細胞は血管内皮細胞また
はマクロファージを包含する。ホモジェネートは、たと
えば心臓、血管、神経または他の組織および細胞からの
ものであり得る。生理学的に活性なNG−アミノアルギニ
ンまたはその塩は１ナノモル−300ミリモルの範囲内に
ある濃度で媒質に添加される。Hereinafter, the in vitro method in this specification will be described. Representative media include cardiac perfusion media, tissue culture media, culture media or purified proteins used with cell or tissue homogenates. The organ to be treated is typically a blood vessel,
Lung or kidney. Intact cells include vascular endothelial cells or macrophages. Homogenates can be, for example, from the heart, blood vessels, nerves or other tissues and cells. Physiologically active NG -aminoarginine or a salt thereof is added to the medium at a concentration in the range of 1 nanomolar to 300 millimolar.

以下本発明を実施例により説明する。 Hereinafter, the present invention will be described with reference to examples.

実施例Ｉ 4.38（20mmol）NG−ニトロ−Ｌ−アルギニン（シグマ
ケミカルズ）を酸化白金0.1gmを含む15％水性酢酸25m
lに溶解した。反応を40ポンド／平方インチH2圧および
室温で約60時間行った。触媒をアルゴン下で濾過により
除去し、濾液を減圧下でロータリー蒸発して油状物にし
た。分析は55％NG−アミノ−Ｌ−アルギニンおよび45％
Ｌ−アルギニンを示した。溶液をNaOHでpH9.5に調節
し、アルギナーゼ5mg（1050国際単位）を添加した。生
成する溶液を一晩中37℃でインキュベートした。この処
理の後、溶液はNG−アミノ−Ｌ−アルギニンおよびＬ−
オルニチンを含んでいたが、Ｌ−アルギニンを含まなか
った。５−10分間100℃に溶液を加熱することによりア
ルギナーゼを変性し、濾過により除去した。フラビアン
酸水溶液（100ml中に6.28gm）をpHが約3.8で、沈澱が生
成しはじめるまで添加した。溶液を一晩中40℃に冷却し
て、生成物の沈澱を完了し、沈澱物を濾過により集めた
（約3.0gm）。固体を1MNaOHの滴下により熱湯（100ml）
に再溶解した。pH3.8へのHCl溶液の添加はNG−アミノ−
Ｌ−アルギニン−フラビアン酸塩の結晶の形成を起し
た。４時間４℃での溶液の冷却後、黄色の結晶を濾過に
より集め、逐次無水アルコールおよびエチルエーテルで
洗浄した。生成物（2.4gm）は、純度の高いNG−アミノ
−Ｌ−アルギニン−フラビアン酸塩（C16H21N7O10Sに対
する計算値:C＝38.2％;H＝4.2％;N＝19.5％。実験値:C
＝38.4％;H＝4.2％;N＝19.5％）であった。モノフラビ
アン酸塩を100℃で水100mlに懸濁し、ダウェックス１
（OH-）（強塩基イオン交換樹脂）10gmを添加した。５
時間100℃で撹拌後、上澄液は透明となり、全ての黄色
のフラビアン酸はダウェックス１樹脂に結合した。樹脂
を濾過により除去し、澄明な濾液を減圧でロータリー蒸
発により濃縮した油状物にした。油状物をエタノール50
mlに溶解し、エタノールを減圧で蒸発した。エタノール
の添加および除去を再三くり返し、次いでエチルエーテ
ル50mlを同様に添加し、除去した。固体状残留物を12時
間P2O5で高真空下で乾燥した。収量は純粋な（99.9＋％
純度）NG−アミノ−Ｌ−アルギニン（出発NG−ニトロ−
Ｌ−アルギニンを基にした35％の収率）1.2mgであっ
た。Example I 4.38 (20 mmol) NG -nitro-L-arginine (Sigma Chemicals) containing 25 g of 15% aqueous acetic acid containing 0.1 gm of platinum oxide
dissolved in l. The reaction was carried out for 40 lbs / square inch H 2 pressure and room temperature for about 60 hours. The catalyst was removed by filtration under argon and the filtrate was rotary evaporated under reduced pressure to an oil. The analysis was 55% NG -amino-L-arginine and 45%
L-arginine was indicated. The solution was adjusted to pH 9.5 with NaOH and 5 mg (1050 IU) of arginase was added. The resulting solution was incubated overnight at 37 ° C. After this treatment, the solution contains NG -amino-L-arginine and L-
It contained ornithine but no L-arginine. Arginase was denatured by heating the solution to 100 ° C. for 5-10 minutes and removed by filtration. An aqueous flavonic acid solution (6.28 gm in 100 ml) was added until the pH was about 3.8 and a precipitate began to form. The solution was cooled to 40 ° C. overnight to complete the precipitation of the product, and the precipitate was collected by filtration (about 3.0 gm). Hot water (100ml) by adding 1M NaOH dropwise
Was re-dissolved. The addition of the HCl solution to pH 3.8 is NG -amino-
The formation of crystals of L-arginine-flavianate occurred. After cooling the solution at 4 ° C. for 4 hours, the yellow crystals were collected by filtration and washed successively with anhydrous alcohol and ethyl ether. Product (2.4gm) is high purity N G - amino -L- arginine - flavianic acid salt (C 16 H 21 N 7 O 10 Calculated for S: C = 38.2%; H = 4.2%; N = 19.5 %, Experimental value: C
= 38.4%; H = 4.2%; N = 19.5%). The monoflavianate is suspended in 100 ml of water at 100 ° C.
(OH -) was added (strong base ion exchange resin) 10 gm. 5
After stirring at 100 ° C. for a time, the supernatant became clear and all yellow flavanic acid bound to the Dowex 1 resin. The resin was removed by filtration, and the clear filtrate was concentrated by rotary evaporation at reduced pressure to a concentrated oil. Oily ethanol 50
and ethanol was evaporated under reduced pressure. The addition and removal of ethanol was repeated three times, and then 50 ml of ethyl ether were similarly added and removed. The solid residue was dried under high vacuum over P 2 O 5 for 12 hours. Yield pure (99.9 +%
Purity) NG -amino-L-arginine (starting NG -nitro-
(35% yield based on L-arginine) 1.2 mg.

実施例II NG−ニトロ−Ｌ−アルギニン（4.38gm）を実施例Ｉに
記載のようにＬ−アルギニンおよびNG−アミノ−Ｌ−ア
ルギニンの混合物に還元し、アルギナーゼで処置した。
アルギナーゼを欠く溶液を乾固するまで蒸発し、乾燥し
た残留物を水10mlに溶解し、生成する溶液をダウェック
ス50（H+）樹脂のカラム（2.5×45cm）に適用した。樹
脂を水（500ml）により、次いで水１リットルと2M水酸
化アンモニウム１リットルとの間に形成した直線状勾配
により洗浄した。25mlの画分を集め、オルニチンおよび
／またはNG−アミノ−Ｌ−アルギニンのそれらの含量に
ついて分析した。純粋なNG−アミノ−Ｌ−アルギニンを
含有している画分をプールし、減圧で乾固するまでロー
タリー蒸発した。半結晶性残留物をP2O5で真空下乾燥し
た。乾燥後、白色の固体（1.5gm）は99.9＋％純度のNG
−アミノ−Ｌ−アルギニンであった。Example II NG -nitro-L-arginine (4.38 gm) was reduced to a mixture of L-arginine and NG -amino-L-arginine as described in Example I and treated with arginase.
The solution lacking arginase was evaporated to dryness, the dried residue was dissolved in 10 ml of water, and the resulting solution was applied to a column (2.5 × 45 cm) of Dowex 50 (H + ) resin. The resin was washed with water (500 ml) followed by a linear gradient formed between 1 liter of water and 1 liter of 2M ammonium hydroxide. 25 ml fractions were collected and analyzed for their content of ornithine and / or NG -amino-L-arginine. Fractions containing pure NG -amino-L-arginine were pooled and rotary evaporated under reduced pressure to dryness. The semi-crystalline residue was dried under vacuum over P 2 O 5 . After drying, a white solid (1.5 gm) was 99.9 +% pure NG
-Amino-L-arginine.

実施例III 体重500gmの雄のハートレイ系モルモットをペントバ
ルビタールナトリウムで麻酔にかけ（50mg/kg腹腔
内）、気管カニューレを挿入する。左頚動脈をカニュー
レ挿入し、生理学的圧力変換器に連結する。血圧の追跡
をフィジオグラフで表す。心搏拡張期の血圧を生理食塩
水、NG−メチル−Ｌ−アルギニン（0.1,1または10mg/kg
体重）またはNG−アミノ−Ｌ−アルギニン（0.1,1また
は10mg/kg体重）の静脈投与によりモニターする。別個
のモルモットを各々の化合物に対して用いる。薬剤投与
５分後、1mg/kgおよび10mg/kgNG−メチル−Ｌ−アルギ
ニンが、それぞれ血圧10および25mmHgを増大したのに対
して生理食塩水および0.1mg/kgNG−メチル−Ｌ−アルギ
ニンは血圧に影響を及ぼさない（アイサカ、ケイ他、前
掲の882頁）。0.1,1および10mg/kg NG−アミノ−Ｌ−
アルギニンの投与５分後、心搏拡張期の血圧は、それぞ
れ10,20および40mmHg増大する。Example III A male Hartley guinea pig weighing 500 gm is anesthetized with sodium pentobarbital (50 mg / kg ip) and a tracheal cannula is inserted. The left carotid artery is cannulated and connected to a physiological pressure transducer. The blood pressure tracking is represented by a physiograph. The blood pressure during diastole is adjusted with saline, NG -methyl-L-arginine (0.1, 1 or 10 mg / kg).
Body weight) or by intravenous administration of NG -amino-L-arginine (0.1, 1 or 10 mg / kg body weight). A separate guinea pig is used for each compound. Drug administration 5 min after, 1 mg / kg and 10 mg / kgN G - methyl -L- arginine, saline and whereas respectively increased blood pressure 10 and 25mmHg 0.1mg / kgN G - methyl -L- arginine It does not affect blood pressure (Aisaka, Kei et al., P. 882, supra). 0.1, 1 and 10 mg / kg NG -amino-L-
Five minutes after administration of arginine, diastolic blood pressure increases by 10, 20, and 40 mmHg, respectively.

実施例IV 血管の弛緩をモルモットの大動脈または肺動脈から取
った分離した輪を用いて好便に研究する。上記の輪への
アセチルコリンの添加はＬ−アルギニンから酸化窒素の
合成を引き起し、生成した酸化窒素は脈管輪の血管緊張
力を制御している平滑筋細胞の弛緩を引き起す。酸化窒
素生成の阻害はアセチルコリンの弛緩作用を減退する。
このイン ビトロ系は過度の血管の弛緩により化学的に
誘発した低血圧症を研究するのに好適なモデルである。Example IV Vascular relaxation is conveniently studied using isolated rings taken from the guinea pig aorta or pulmonary artery. Addition of acetylcholine to the annulus causes the synthesis of nitric oxide from L-arginine, and the nitric oxide produced causes the relaxation of smooth muscle cells that control vascular tone of the vascular ring. Inhibition of nitric oxide production reduces the relaxing action of acetylcholine.
This in vitro system is a suitable model to study hypotension chemically induced by excessive relaxation of blood vessels.

モルモットの大動脈および肺動脈の輪のアセチルコリ
ン仲介弛緩に及ぼすNG−アミノ−Ｌ−アルギニンの影響
をサクマ、アイ他、プロシーディングス・オブ・ザ・ナ
ショナル・アカデミー・オブ・サイエンス・ユーエスエ
ー第85巻、8664−8667頁（1988年）に記載した方法によ
り記載通り測定した。アセチルコリンの用量は10-8−10
-5Mの範囲である。NG−アミノ−Ｌ−アルギニンを0.3マ
イクロモル、10マイクロモルおよび30マイクロモルの用
量で添加した。NG−アミノ−Ｌ−アルギニンの無い場
合、10-8,10-7,10-6および10-5Mの用量でのアセチルコ
リンは、それぞれ約10％,23％,43％および58％の弛緩を
引き起した。３マイクロモルのNG−アミノ−Ｌ−アルギ
ニンが有る場合での同じ実験は０％,0％,5％および19％
の弛緩を得た。NG−アミノ−Ｌ−アルギニンの濃度を10
マイクロモルまたは30マイクロモルに増大した場合、10
-6Mアセチルコリンにより誘発した弛緩の範囲は１％未
満で、10-5Mアセチルコリンにより起った範囲は10％未
満であった。それより低用量のアセチルコリンは10マイ
クロモルまたは30マイクロモルNG−アミノ−Ｌ−アルギ
ニンのある場合に弛緩を起さなかった。結果は、アセチ
ルコリン支配の血管の弛緩が大部分Ｌ−アルギニンから
の酸化窒素生成によるもので、NG−アミノ−Ｌ−アルギ
ニンによって阻害されることを示す。上記の研究がイン
ビボの低血圧症に対する好適なモデルであるから、結
果はまたは酸化窒素仲介血管の弛緩による低血圧を上昇
させることについてのNG−アミノ−Ｌ−アルギニンのイ
ン ビボの有用性を示す。The effect of NG -amino-L-arginine on acetylcholine-mediated relaxation of the guinea pig aortic and pulmonary artery rings was described by Sakuma, Ai et al., Proceedings of the National Academy of Sciences USA 85: 8664. -Measured as described by the method described on page 8667 (1988). Acetylcholine dose is 10 -8 -10
-5 M range. NG -amino-L-arginine was added at 0.3, 10 and 30 micromolar doses. In the absence of NG -amino-L-arginine, acetylcholine at doses of 10 -8 , 10 -7 , 10 -6 and 10 -5 M relax about 10%, 23%, 43% and 58%, respectively. Caused. The same experiment with 3 micromolar of NG -amino-L-arginine shows 0%, 0%, 5% and 19%
Was obtained. NG -amino-L-arginine concentration of 10
When increased to micromolar or 30 micromolar, 10
The extent of relaxation induced by -6 M acetylcholine was less than 1%, and the extent caused by 10 -5 M acetylcholine was less than 10%. Lower doses of acetylcholine did not cause relaxation in the presence of 10 or 30 micromolar NG -amino-L-arginine. The results show that acetylcholine-controlled vasorelaxation is largely due to nitric oxide production from L-arginine and is inhibited by NG -amino-L-arginine. Since the above study is a preferred model for in vivo hypotension, the results or the utility of NG -amino-L-arginine in vivo for increasing hypotension by nitric oxide-mediated vasorelaxation Is shown.

同じ研究において、血管輪弛緩の同程度の阻害を起す
のにNG−アミノ−Ｌ−アルギニンの上にした用量よりも
５−10倍高い濃度のNG−メチル−Ｌ−アルギニンが必要
であった。In the same study, 5-10 fold higher concentrations of NG -methyl-L-arginine were required to produce the same degree of inhibition of vascular ring relaxation than doses over NG -amino-L-arginine. Was.

等モル量のNG−ニトロ−D,L−アルギニンを実施例Ｉ
またはIIにおけるNG−ニトロ−Ｌ−アルギニンに置換す
る場合、純粋なNG−アミノ−D,L−アルギニンを得る。Example I was prepared by equimolar amounts of NG -nitro-D, L-arginine.
Alternatively, when substituting for NG -nitro-L-arginine in II, pure NG -amino-D, L-arginine is obtained.

実施例IIIおよびIVにおいて、NG−アミノ−D,L−アル
ギニンを用量または濃度を２倍にしてNG−アミノ−Ｌ−
アルギニンに置換する場合、実質的に心搏拡張期の血圧
増大の同等の結果および血管輪の弛緩の阻害を得る。In Examples III and IV, NG -amino-D, L-arginine was dosed or doubled to give NG -amino-L-arginine.
When substituting for arginine, one obtains substantially the same result of increased blood pressure during diastole and an inhibition of relaxation of the vascular ring.

本発明の具体例の多くの変形は当業者に明らかであ
る。すなわち、本発明の実施態様は請求範囲により定義
される。Many modifications of the embodiments of the invention will be apparent to those skilled in the art. That is, the embodiments of the present invention are defined by the claims.

───────────────────────────────────────────────────── フロントページの続き (56)参考文献 Ａｃｔａ Ｃｈｉｍｉｃａ Ａｃａｄ ｅｍｉａｅ Ｓｃｉｅｎｔｉａｒｍ Ｈ ｕｎｇａｒｉｃａｅ，Ｔｏｍｕｓ 第85 巻第３号，第327〜332頁 （1975) Ｂｉｏｃｈｅｍｉｃａｌ Ａｎｄ Ｂ ｉｏｐｈｙｓｉｃａｌ Ｒｅｓｅａｒｃ ｈ Ｃｏｍｍｕｎｉｃａｔｉｏｎｓ 第 160巻第２号，第881〜886頁 （1989) ────────────────────────────────────────────────── ─── Continuation of the front page (56) References Acta Chimica Acad emiia Scientific Hungaricae, Tomus Vol. 85, No. 3, 327-332 (1975) Biochemical And Biophysical Communication No. 160 881-886 (1989)

Claims (1)

(57)【特許請求の範囲】(57) [Claims] 【請求項１】NG−アミノ−Ｌ−アルギニンまたはその医
薬的に許容可能な塩を有効成分とする、アルギニンから
の酸化窒素合成阻止剤。1. An agent for inhibiting the synthesis of nitric oxide from arginine, comprising NG -amino-L-arginine or a pharmaceutically acceptable salt thereof as an active ingredient.
JP2510618A 1989-09-13 1990-07-30 Separation of aminoarginine and uses to block nitric oxide production in the body Expired - Fee Related JP2728148B2 (en)

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